Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) is a category C priority pathogen with a high fatality rate that retains the potential to cause a pandemic. We aim to elucidate the mechanisms by which such enveloped viruses mediate membrane fusion during the process of entry. Inhibitors of entry will be sought through targeted and general screening of compound libraries. This work will have important consequences on the development of treatments for SARS-CoV, as well as understanding how entry occurs for SARS-CoV and other emerging viral infections with similar properties, such as ebolavirus. Furthermore, we will identify novel targets for the development of new antivirals. We have previously determined that the spike envelope glycoprotein of SARS-CoV (termed S) requires proteolytic processing by cathepsin L, and likely other proteases, in order to facilitate its membrane fusion potential. Uniquely, interactions with obligate receptor (Angiotensin Converting Enzyme-2, ACE2) are initially required before proteolysis. We hypothesize that ACE2 interaction induces conformational changes that not only exposes cryptic cleavage sites, but also initiates the cascade of rearrangements within S necessary to mediate membrane fusion. However, conformational constraints prevent the completion of these rearrangements. Hence, proteolysis is required in order to release these restraints. We base these hypotheses on the observations that: (1) broad spectrum and specific protease inhibitors able to block cathepsin L efficiently inhibit SARS-CoV entry; (2) recombinant cathepsin L, as well as a number of serine proteases such as trypsin, are able to successfully mediate processing of S leading to membrane fusion; (3) an elevated temperature-dependent step subsequent to receptor engagement, but prior to proteolysis is required for efficient membrane fusion. Residual SARS-CoV infection of cathepsin L- deficient cells is observed. Thus, we postulate that other endosomal proteases can mediate these effects. Furthermore, extracellular or surface proteases in tissue such as the lung may be able to mediate cell surface activation of S. We aim to identify proteases able to mediate infection and the mechanisms by which this is achieved. Inhibitors of SARS-CoV mediated infection will be sought, by specifically targeting cellular proteases, as well as by conducting a broader screen of viral entry. The specific aims are to: 1. Identify endosomal proteases able to mediate SARS-CoV infection and characterize the sites and mode of cleavage. Inhibitors of this proteolytic activity will be characterized as potential lead therapeutics. 2. Characterize the triggers of S- mediated membrane fusion, and hence uncover new targets for inhibitors. 3. Identify small molecule inhibitors of SARS-CoV S-mediated entry, and characterize their mode of action. This will identify both lead compounds for antivirals directed against SARS-CoV, highlight targets for inhibitors of viral entry and provide useful reagents for aims 1 and 2. The high mortality rate and large animal reservoir of SARS-associated viruses in Asian bats, coupled with a lack of proven therapeutic or prophylactic reagents, raises concern about SARS-CoV becoming a significant worldwide public health hazard. We propose the targeted and general screening of extensive small molecule libraries in order to identify inhibitors of viral entry that can be developed as SARS-CoV antivirals. Candidate inhibitors will be developed as lead compounds for the prophylactic and therapeutic treatment of this serious respiratory infection.